Double-labelled HIV-1 particles for study of virus–cell interaction

Abstract

Human immunodeficiency virus (HIV) delivers its genome to a host cell through fusion of the viral envelope with a cellular membrane. While the viral and cellular proteins involved in entry have been analyzed in detail, the dynamics of virus-cell fusion are largely unknown. Single virus tracing (SVT) provides the unique opportunity to visualize viral particles in real time allowing direct observation of the dynamics of this stochastic process. For this purpose, we developed a double-coloured HIV derivative carrying a green fluorescent label attached to the viral matrix protein combined with a red label fused to the viral Vpr protein designed to distinguish between complete virions and subviral particles lacking MA after membrane fusion. We present here a detailed characterization of this novel tool together with exemplary live cell imaging studies, demonstrating its suitability for real-time analyses of HIV-cell interaction.

HIV-1–cellular interactions analyzed by single virus tracing

“…293T cells cultivated in DMEM with 10% fetal calf serum and antibiotics were transfected with a mixture of plasmids; pCHIV, pCHIV.eGFP and pmRFP-Vpr (Lampe et al 2007) in a molar ratio of 1:1:0.8 by calcium phosphate precipitation. At 44 h after transfection, medium was harvested and cleared, (Lampe et al 2007) and VLPs were concentrated by ultracentrifugation through a 20% (w/w) sucrose cushion.…”

“…At 44 h after transfection, medium was harvested and cleared, (Lampe et al 2007) and VLPs were concentrated by ultracentrifugation through a 20% (w/w) sucrose cushion. Particles were resuspended in ice cold PBS, 10% fetal calf serum and frozen.…”

“…The average particle size is comparable to the HIV-1 particle diameter of 145 ± 25 nm previously determined by cyro-electron microscopy (Briggs et al 2003), indicating that the virus preparations provide a relatively homogeneous population of complete, nonaggregated single VLPs. A dual-color HIV-1 derivative, which carries mRFP1 fused to the HIV-1 accessory protein Vpr (Vpr.mRFP) in addition to MA.eGFP, was also developed and characterized (Lampe et al 2007). These particles are designed to distinguish between complete, dual-colored VLPs and subviral complexes entering the cytoplasm, which should lose most of the MA.eGFP label upon membrane fusion and retain the Vpr.mRFP1 label.…”

“…For a detailed investigation of HIV-cell interactions by SVT, we have generated and characterized entry-competent single-as well as double-fluorescently labeled HIV-1 derivatives (Lampe et al 2007;Müller et al 2004). The dual-labeled virus like particles (VLPs) carry two differently colored fluorescent labels at two viral proteins and were designed to distinguish between complete virions and subviral particles resulting from membrane fusion.…”

“…The dual-labeled virus like particles (VLPs) carry two differently colored fluorescent labels at two viral proteins and were designed to distinguish between complete virions and subviral particles resulting from membrane fusion. In our initial live-cell imaging studies using these VLPs, we had observed different types of particle behavior: free diffusion, brief touches with the plasma membrane, as well as a significant percentage of particles that displayed a rapid, presumably non-productive, immobilization at the plasma membrane in an apparently heparan sulfate dependent manner (Lampe et al 2007). Heparan sulfate has been reported to be involved in HIV-1 cell attachment and to be of functional importance in some types of host cells, but the overall role of heparan sulfate for HIV-1 infection is not entirely clear (Bobardt et al 2003;Moulard et al 2000;Parren et al 1998;Ugolini et al 1999;Zhang et al 2002).…”